The task of the present invention is to disclose a microscope that enables variable adjustment of the penetration depth of illumination light during evanescent illumination of a sample.
This task is solved by a microscope wherein an adjustment mechanism is provided with which the polarization of the illumination light beam may be changed.
A further task of the present invention is to disclose an illumination module for a microscope that enables illumination of a microscopic sample during evanescent sample of illumination with adjustable penetration depth.
The further task is solved by an illumination module wherein the illumination module can be coupled to a microscope for the purpose of evanescent illumination of a sample, and wherein an adjustment mechanism is provided with which the polarization of the illumination light beam may be changed.
It has been recognized, according to the invention, that the penetration depth (and the light power) of an evanescent illumination field in a sample is dependent on the polarization of the illumination light that strikes the cover glass-sample interface or the sample holder-sample interface, respectively.
By means of the microscope according to the invention or the illumination module according to the invention, respectively, the orientation of sample objects (such as molecules, cell components, etc.) may also advantageously be determined, as well as the corresponding isotropy of the refracted space.
In a preferred variant, the microscope exhibits an objective with an objective pupil, whereby the illumination light beam for evanescently illuminating the sample exhibits a focus in the area of the objective pupil. Preferably, an adjustable beam deflector is provided with which the position of the focus within the objective pupil may be moved. This creates an additional possibility for changing the penetration depth.
It has also been recognized, according to the invention, that in addition to the polarization, the penetration depth of an evanescent illumination field in a sample is dependent on the angle at which total reflection occurs at the cover glass interface or at the sample holder interface, respectively. This angle is directly correlated with the angle relative to the optical axis at which the illumination light beam provided for evanescent sample illumination exits the objective via the front lens. This angle, in turn, is dependent on the distance to the optical axis at which the illumination light beam passes through the rear focal plane of the objective (pupil). In order to have available a largely parallel illumination light beam for evanescently illuminating a sample, the illumination light beam must exhibit a focus in the rear focal plane of the objective. Finally, the distance of the focus to the optical axis of the objective determines said angle, and thereby the penetration depth of the evanescent field in the sample that is to be tested.
In a preferred embodiment of the microscope according to the invention, an adjustable beam deflector is arranged in the beam path of the illumination light beam. The beam deflector preferably comprises at least one galvanometric mirror. In order to position the focus at any given location within the objective pupil, the beam deflector preferably comprises two galvanometric mirrors, which cause deflection of the illumination light beam in different lateral directions (e.g., x- and y-direction). The beam deflector may also comprise rotatable or tippable prisms and/or rotatable or tippable mirrors. The use of acousto-optical or electro-optical deflection elements can also be envisioned.
The adjustment mechanism with which the polarization of the illumination light beam may be adjusted preferably comprises a phase plate, preferably a rotatable λ/2 plate—preferably motorized. The adjustment mechanism may also comprise a Faraday rotator and/or a Pockels cell and/or a double-refractive material and/or a liquid crystal cell.
In a particularly preferred embodiment of the microscope or of the illumination module, respectively, the adjustment mechanism is the control element of a regulator, which adjusts the polarization according to settings input by the user.
In a very particularly preferred variant, storage memory is provided in which—preferably sample-specific—polarization settings are stored to achieve different penetration depths. In this manner, adjustment, according to the invention, is quantifiable and reproducible.
A control mechanism is preferably provided to measure and/or monitor the polarization of the illumination light beam. In a preferred variant, the control mechanism is the measuring element of the regulator.
Advantageously, the control mechanism may comprise a beam splitter that outcouples the measuring light from the illumination light beam. In an advantageous embodiment, the control element comprises at least one detector that detects the light power of at least a part of the measuring light.
A particularly preferred variant is one in which the control mechanism comprises at least one polarization analyzer, which is preferably arranged in the beam path of the measuring light before the minimum of one detector. It may, for example, be a polarization foil, a double-refractive prism (e.g., a Glan-Thomson prism), or a polarization beam splitter, which may, for example, be implemented as a cube.
In a particular embodiment of the microscope or of the illumination module, respectively, the polarization beam splitter splits the measuring light into an s-polarized measuring beam and a p-polarized measuring beam. Preferably, two detectors are provided, of which one detector receives the s-polarized measuring beam and the other the p-polarized measuring beam. In this manner, precise conclusions may be drawn from the light power measured by both detectors regarding the polarization of the illumination light beam. Preferably, a processing module is provided with which the measurement data are processed. The processing module may also be a component of the regulator.
At least the light source and the adjustment mechanism are preferably integrated into an illumination module that may be detachably coupled to a microscope stand or to an already existent microscope. The illumination module preferably also comprises the control mechanism. A bayonet coupling is preferably provided.
The microscope preferably comprises a camera and/or a CCD element and/or an EMCCD element for the purpose of imaging.
In a preferred variant, a power adjustment mechanism is provided to change the light power of the illumination light beam. This may, for example, be a mechanical beam attenuator, an LCD module, or an electro-optical—or acousto-optical—component (e.g., AOTF).
The microscope preferably comprises a scanning microscope, in particular a confocal scanning microscope.
In a particular variant, at least the light source and the adjustment mechanism are integrated into an illumination module, which preferably may be coupled to a microscope and/or to a microscope stand.
The illumination module according to the invention provides the advantage that it may be coupled as a retrofit to an already existent microscope or microscope stand.